Epidemic viral infections are responsible for significant worldwide loss of life and income in human illnesses ranging from the common cold to life-threatening influenza, West Nile and HIV infections. Timely detection, diagnosis and treatment are key in limiting spread of disease in epidemic, pandemic and epizootic settings. Rapid screening and diagnostic methods are particularly useful in reducing patient suffering and population risk. Similarly, therapeutic agents that rapidly inhibit viral assembly and propagation are particularly useful in treatment regimens.
Influenza A has emerged recently as a potential significant risk to human populations. Avian strains have crossed into humans and there is growing evidence that human to human spread may soon occur1. Examples of the impact of avian influenza strains on human populations is provided by the recent emergence of highly virulent strains of avian influenza H5N1 (bird flu) where approximately 50% of infected individuals (42 people) succumbed and food shortages resulted from slaughter of millions of birds in China, Indonesia and Vietnam. Tracking the potential for epidemic, the World Health Organization considered raising the global threat level to 4 or 5 (on a scale of six) in July of 2005. One opinion leader recently expressed in press that with avian influenza—“detection, surveillance, prevention and therapy” . . . (is) . . . “a race against time”1. Since avian strains have rarely been isolated from humans and mortality rates in humans are high, it seems likely that immunity in the worldwide population is virtually non-existent. Thus, the opportunity exists for a worldwide pandemic. For comparison, in 1918 a global influenza epidemic resulted in an estimated 20-40 million deaths. With increased population density today, higher mortality is likely.
Virology test methods for detection and confirmation of influenza A infection in a virus-secure reference laboratory, e.g., satisfying requirements for Containment Group 4 pathogens, are time consuming, high-risk and laborious, i.e., involving 4-7 days isolation of the virus in embryonated eggs; harvesting allantoic fluids from dead or dying embryos; testing the fluid in hemagglutination and hemagglutination inhibition tests, immunodiffusion; and, eventual subtyping of the virus in the fluid by hemagglutinin and neuraminidase in overnight immunodiffusion assays using specially prepared monospecific antisera. Present subtyping involves identifying each of 16 different possible viral hemagglutinin proteins in combination with 9 different possible viral neuraminidase proteins. Unfortunately, since only a few pathogenic strains of influenza A are of economic and health concern at any point in time, much of this time-consuming effort may be unnecessary and wasted.
Current rapid immunodiagnostic tests for influenza antigens like “Binax NOW FluA and FluB™” (Binax, Inc., Porltand, Me.), “Directigen Flu A+B™” (Becton Dickinson, Franklin Lakes, N.J.), “Flu OIA™” (Biostar Inc., Boulder, Colo.), “Quick Vue™” (Quidel, Sand Diego, Calif.), “Influ AB Quick™” (Denka Sieken Co., Ltd., Japan) and “Xpect Flu A & B” (Remel Inc., Lenexa, Kans.), can reportedly either detect influenza A or distinguish between Influenza A and B, but importantly, not between different influenza A subtypes or between pathogenic and non-pathogenic strains of influenza A. The complexity of the test formats may require special training. In addition, significant amounts of virion particles are commonly required to obtain a positive test result, limiting their use to a short window of time when virus shedding is at its highest levels. Assay sensitivity is also variable with up to 20% false negative test results in certain assays being of significant current concern (e.g., see “WHO recommendations on the use of rapid testing for influenza diagnosis”, July 2005). Recent introduction of reverse-transcriptase PCR-based diagnostics (RT-PCR) for confirming influenza A virus have resulted in important advances in capabilities36, but are laborious and require highly trained personnel making on-site or field-testing difficult. Because of the relative inefficiency of the reverse transcriptase enzyme, significant amounts of virus (e.g., 104 virion particles) and as many as 20 primers may be required to effectively detect viral RNA. Despite these significant obstacles, in reference laboratory RT-PCR influenza A testing high levels of proficiency have recently been recorded between 12 different participating test laboratories in the US, Canada and Hong Kong36. Using RT-PCR and HA primers, Lee et al.37 described quantitative discrimination between H5 and H7 subtypes of virus. Munch et al.38 report similar possible differential specificity in RT-PCR using NP primers. Unfortunately, RT-PCR is not easily adapted to high throughput screening of subjects in an epidemic setting or to field uses in an agricultural or point-of-care setting.
Additionally, the complexity, diversity and rapid emergence of new influenza strains has made diagnosis of high risk strains difficult, and therefore rapid response is at present nearly impossible. For epidemiologists, diversity resulting from high mutation rates and genetic reassortment make it difficult to anticipate where new strains may originate and respond with the timely introduction of new diagnostic primers for PCR. As a result, (at present) the diversity of influenza dictates the necessity of multiplex PCR approaches.
Avian influenza virus (H5N1) is believed to be evolving by both mutation and segmental reassortment with influenza viruses in aquatic wildfowl2,3. Highly pathogenic disease in “sick” birds may vary from sudden death with few overt signs of disease to a more characteristic disease with respiratory signs, excessive lacrimation, sinusitis, edema of the head, cyanosis of the unfeathered skin and diarrhea, i.e., the diagnostic signs of “sick” employed by OIE in their health guidelines (Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 5th edition, 2004, World Organization for Animal Health). In infected birds influenza A virus is shed in just 2-3 days4,5. Given the high mortality rates in humans, rapid detection is essential to isolate infected avian and human subjects and protect human populations. In the field, human cases of bird flu have historically originated in regions of South East Asia that lacks easy access to sophisticated diagnostic test equipment, virus-secure reference BL4 laboratories and methods. Thus, assessing population risk at an individual patient level is presently highly problematic. In other objects, the invention offers solutions to these problems.
Rapid diagnostic testing, needed to support agriculture and the public health, is proving to be challenging—i.e., for either serological detection of anti-viral host responses (antibody) or identification of viral proteins (antigens) in samples. Testing for influenza A subtypes is also complicated by: (i) the scope of epidemiological and the public health needs, i.e., potential needs for viral detection in environmental samples and in infected livestock, (e.g. swine flu), poultry (e.g. avian flu) and humans (e.g. bird flu); and, (ii) the wide range of possible test samples which may include serum, nasopharyngeal, throat gargle, nasal or laryngeal samples (human); and, cloacal, feces and tracheal samples (bird). Since high risk viruses tend to spread rapidly, speed is of the essence. High affinity specific binding reagents are clearly key and required. In other objects, the invention solves these key needs.
Classical influenza serological testing (for antibody) by hemagglutination-inhibition (HI) is relatively simple, but in agricultural practice these tests are relatively insensitive for detecting avian antibody responses following either vaccination or natural infection as serum antibody tends to fall rapidly after infection. Under optimal conditions Xu et al.39 e.g. recently described a latex agglutination test, i.e., using complete heat inactivated vaccine virus and serum from vaccinated birds. The latter HI-test reportedly had 88% sensitivity (12% false negatives) and 98% specificity, in this case, false negative rates too high for agricultural or public health detection of such dangerous viral pathogens. Similarly, using avian field samples in China, Jin et al.4 recently described potential uses of a recombinant influenza NP antigen in ELISA assays. These investigators observed that virus shedding began at days 2, but titers of anti-viral antibodies were most significant at 2 weeks. Unfortunately, the latter “lag” before detection of infected animals is unacceptable in the current worldwide crisis. Demonstrating a further possible complication, data in the latter studies showed that low doses of virus generated only very low titers of antibody, i.e., suggesting that subclinical infections might go undetected.
Present limitations in routine diagnostic methods for flu, i.e., Influenza B, were noted in data published by Steininger et al.41. In the latter studies, different test methods were employed to detect a standard influenza A stock virus preparation; and, with the following findings: namely, rapid enzyme-based assays were about 1000-fold less sensitive than detection by conventional virus isolation methods; which were, in turn, about 1000-fold less sensitive than RT-PCR. Despite the latter gross quantitative limitations in sensitivity, the ELISA still correctly identified 62% of positive samples and 88% for samples obtained from patients younger than 5 yrs. of age with Influenza B (flu). As an example of the impact that poor samples can have on assay performance, commercially viable flu tests were assayed for their sensitivity in detecting viral antigen in nasopharyngeal samples of experimentally infected volunteers. The reported results suggest that assay sensitivity was about 60% for the Directigen flu test43 (Becton Dickinson); and, in the range of 48-100% for the flu optical immunoassay (FLU OIA; ThermoBioStar/Biota)44. Importantly, (despite the obvious limitations of the latter tests), Sharma et al.45 reported that rapid confirmation of influenza virus type A infection: (i) decreased irrelevant laboratory testing, e.g. urinalysis and wbc testing, as well as, (ii) inappropriate antibiotic use in febrile infants and toddlers. Thus, a relatively poor sensitivity in these screening assays was still useful in clinical practice because the assay correctly identified those patients who needed additional follow-up. Clearly, for non-reference lab uses, improvements in user friendliness, speed, discrimination and absolute quantitative sensitivity are needed, i.e., even for routine flu testing. Similarly, routine flu testing is not particularly helpful in suggesting how one may achieve a method with the requisite assay performance needed to test for high risk strains of influenza A in patient samples.
Emergent virulence factors in H5N1 and H7 avian influenza A viruses and the panzooic spread of H9N2 influenza virus and their known interactions with mammalian host factors have been reviewed5. Among the proteins encoded by virulent avian strains of influenza, NS1 (non-structural protein-1) is expressed early in infected cells, but unlike HA and NA, it is not virion associated and is expressed only as an intracellular protein. NS1 is encoded by genome segment 8 and is a viral regulatory factor enhancing translation of viral mRNA; interfering with maturation and transport of host cell mRNA6; binding poly(A) tails of host mRNA; altering intrinsic small interfering RNA (siRNA) control of host cell gene expression7; preventing ds-RNA induction of antiviral protein kinase R; inhibiting induction8 of, and antagonizing9,10 anti-viral action of interferon α/β (IFN-α/β); and, stimulating production of pro-inflammatory cytokines by macrophages11 and dendritic cells12. The roles of INF-α/β signaling in innate and adaptive immune responses and pathogenesis has recently been reviewed.13 
Distribution of NS1 protein in infected cells suggest preferential nuclear localization, i.e., but with lesser amounts in cytoplasmic, ribosomal and polysomal fractions22-24. NS1 protein of the highly virulent avian H5N1 strain apparently suppresses interferon responses of human cells in vitro25. Certain mechanistic studies suggest that carboxyl terminal deletions in NS1, may attenuate in vivo virulence of wild-type A/Swine/Texas/4199-2/98 (TX/98) virus26, as well as, equine influenza virus27. Interestingly, Influenza A lacking the NS1 gene seems to replicates best in interferon-deficient cell lines28, suggesting to the authors that NS1 inhibition of INF-α/β may be necessary for efficient viral propagation. In addition, reassortment of the high-virulence H5N1-NS1 gene into the lower virulence H1N1-A strain reportedly reduced lung clearance rates of the hybrid virus, and also resulted in increased levels of inflammatory cytokines29. Tumpey et al.40 reported that detecting anti-NS1 antibodies may be useful in distinguishing vaccinated from infected poultry, i.e., because NS1 is only expressed in infected cells not in inactivated gradient purified vaccine virus. Unfortunately, the latter antibody-based serological test methods suffer from the same general problems identified above in regard to HI tests: namely, low sensitivity and inability to detect virus prior to virus shedding and potential spread of infection.
Using the H7N3 strain, Cattoli et al.42 reportedly evaluated the timing, specificity and sensitivity of detection of virus in tracheal samples from experimentally and naturally infected turkeys, i.e., in antigen-capture ELISA, RT-PCR and a real-time RT-PCR, (i.e., the later two tests targeting the M gene). Under the latter relatively controlled laboratory conditions, virus was detectable with good specificity and sensitivity as early as 3-5 days post-infection. They concluded that it should be theoretically possible to detect, at least this particular avian virus and perhaps other more highly virulent avian strains at day 3 to 5 of infection provided there were sufficient assay sensitivity.
Thus, there remains a significant need in the medical arts for improved, inexpensive, rapid, accurate and discriminatory methods capable of detecting the particular strains of pathogenic viruses most often involved in generating medically important diseases. There is also a special need for simple assay methodologies that can be routinely used by relatively untrained individuals in underdeveloped nations, markets, clinics, doctor's and veterinary offices, schools and food processing plants where resources may be limited and sophisticated lab equipment not widely available. In view of the worldwide threat posed by the spread of new Influenza A variants, there is a need in the clinical arts for new and improved anti-viral medicinal agents. This invention meets these needs.